Activation of resting T lymphocytes through the T cell antigen receptor (TCR) and its co-receptors is initiated by rapid but transient phosphorylation of several regulatory proteins or enzymes on tyrosine residues. At least six protein tyrosine kinases (PTKs) belonging to the Src, Syk, Csk and Tec families are known to be involved in the earliest events induced by these receptors. In contrast, only one protein tyrosine phosphatase (PTPase), CD45, has so far been found to be important in these early signaling events. It is clear that many more PTPases must be involved in a critical manner both in maintaining the resting state of the signaling machinery and in the active transmission of signals. This proposal focuses on one of these PTPases, the low Mr PTPase, LMPTP. Specific Aim 1 attempts to clarify the involvement, importance and regulation of LMPTP in TCR-plus CD28-induced T cell activation. We will focus on our preliminary finding that LMPTP affects the activation of the Zap-70 PTK and the mitogen-activate protein kinase (MAPK) cascade in a positive manner utilizing a number of rapid co-transfection/reporter assays that we have developed. Positive findings will be analyzed in more detail using stably transfected cell lines. Further experiments address the connection between the receptors and LMPTP and our finding that LMPTP is regulated by phosphorylation at Y131 and Y132 and seems to form autocatalytic homodimers. Immunofluorescence experiments also suggest that LMPTP is located at or near the plasma membrane. The biological relevance of all these findings will be evaluated by site-directed mutagenesis. Specific Aim 2 concentrates on identifying the physiological substrate(s) of LMPTP in resting or activated T cells. First, we will build on our finding that LMPTP affects the Zap PTK and the MAPK pathway and examine the regulators of these enzymes and other potential mechanisms. Second, we will use two unbiased approaches to find LMPTP substrates using the substrate-binding properties of the D129A mutant of LMPTP. Once a substrate has been found, further experiments will identify the specific sites of dephosphorylation by LMPTP, and the role of these sites for the function of the substrate in T cell activation. We anticipate that the results obtained in this study will enable us to better understand the molecular events that follow TCR plus CD28 triggering and govern the onset of T cell proliferation. This will not only benefit our understanding of the initiation of an immune response, but also the normal regulation of proliferation in other cell types as well. This information, in turn, is crucial for the study of deregulated and malignant proliferation of cells, e.g., in T cell lymphomas and other malignant diseases.